Embryonic or early postnatal exposure to environmental compounds such as endocrine disruptors have been shown to promote abnormal development and disease in a variety of species, including humans [135, 136, 137, 138]. Although a large number of environmental factors, such as smoking and caloric restriction [139, 140], and synthetic compounds ranging from plastics to pesticides [141, 142], can promote adult onset disease and abnormalities, the mechanisms of action of these factors are largely unknown.
“Genetic imprinting”, suggests that some genes “remember” the sex of the parent they came from and that this “parental memory” regulates their activity. This regulation can take different forms, but usually one allele is silenced so that only one parental copy is active. For some imprinted genes only the paternal allele is active, while for other imprinted genes only the maternal allele is active. This differential gene activity dependent on the sex of the parent that transmitted the copy of the gene is called “genetic imprinting.” In order for genetic imprinting to occur, some kind of molecular mark must exist to distinguish a maternal allele from a paternal allele. This molecular marker is “epigenetic,” meaning that it does not directly modify the DNA nucleotide sequence but causes a heritable change of gene activity. Epigenetic modifications act like the “font” of the primary basesequence “text,” strongly affecting the genetic message.
A number of human diseases are the result of abnormal epigenetic (i.e. DNA methylation) programming including Angelman's syndrome, Beckwin-Wiedemann syndrome and Prader-Willi syndrome [143, 144, 145]. Alterations in the DNA methylation patterns of imprinted genes have also been shown to promote the development of disease [146]. Potential epigenetic abnormalities in children from in vitro fertilization (i.e. intracytoplasmic sperm injection) have been identified [147]. A study of monozygotic twins suggested environmental epigenetic effects on disease [148]. Therefore, numerous studies and clinical conditions suggest epigenetics may be a critical factor in disease etiology.
Transgenerational effects of irradiation, chemical treatments (e.g. chemotherapy) and environmental toxicants such as endocrine disruptors have been observed over the past decade. The majority of transgenerational observations are simply the effects of the agent on the gestating mother and subsequent actions on the offspring associated with the F1 generation [1-3]. Transgenerational effects for multiple generations have not been as thoroughly studied and require transmission through the germ-line. The ability of an external agent to induce a transgenerational effect may be through an epigenetic phenomenon involving DNA methylation or stable chromosomal alterations [4-7]. Transgenerational effects of irradiation were the first to be identified and some have been shown to be transmitted through the germ-line to multiple generations [4-6]. These are often associated with mutagenesis and tumor formations in subsequent generations. The treatment of cancers with chemotherapeutics also has been shown to cause transgenerational effects [8-10], but the impact on multiple generations has not been thoroughly investigated. Recently, nutritional effects on the F1 generation have been observed [11], but none have been shown to be transgenerational. Environmental toxicants such as endocrine disruptors have also been shown to influence the F1 generation after parental exposure [9, 12-14], but few studies have demonstrated transgenerational effects on multiple generations. However, the potential impact of such transgenerational effects of endocrine disruptors has been discussed [15].
Epigenetic alterations that lead to transgenerational transmission of specific genetic traits or molecular events (e.g. imprinting) have recently been identified [16-19]. These observations have lead to the conclusion that a re-programming through altered methylation state of the germ-line is responsible. The impact this has on human health and evolutionary importance is significant [16, 17]. Recent investigations of the methylation state of the primordial germ cells (PGC) has indicated that as PGC migrate down the genital ridge a de-methylation (i.e. erasure of methylation) starts, and upon colonization in the early gonad a complete de-methylation is achieved [20-22]. This has been primarily observed through the analysis of specific imprinted genes [23]. During the period of sex determination in the gonad the germ cells undergo a re-methylation involving a sex specific determination of the germ cells. Although the de-methylation may not require the gonad somatic cells [21], the re-methylation of the germ-line appears to be dependent on association with the somatic cells in the gonads [20, 22].
Many reports have suggested that environmental endocrine disruptors, which act to mimic estrogens or act as anti-estrogens or anti-androgens, are detrimental to reproduction, and may be the factors responsible for abnormalities, such as a decrease in sperm count, an increase in testicular cancer [24, 25], and an increase in abnormalities in sex determination in many species [26]. Examples of the environmental endocrine disruptors that have been targeted for adverse effects on reproductive systems in humans and other animals are pesticides (e.g. methoxychlor), fungicides (e.g. vinclozolin), a range of xenoestrogens and certain phthalates. Most of these chemicals are ubiquitous in the environment and both humans and other animals are exposed to them daily. Many of these compounds and endocrine disruptors can be metabolized into both estrogenic and anti-androgenic activities [27]. In the current study, methoxychlor and vinclozolin are used as model endocrine disruptors [28] and has both estrogenic and anti-androgenic metabolites [27].
Many environmental endocrine disruptors are weakly estrogenic and elicit their actions through the estrogen receptors. The two mammalian receptors for estrogen (ER-α and ER-β) are widely distributed throughout the reproductive tract and during fetal gonad development [29, 30]. ER-β is present in higher concentrations within the fetal testis and ovary while ER-α is present mainly within the uterus [31, 32]. During fetal testis development ER-β is first expressed in Sertoli and myoid cells after seminiferous cord formation [33]. In rats ER-β has also been localized to pre-spermatogonia, which may explain the proliferative actions of estrogen on early postnatal gonocyte cultures [34]. The importance of ER-α was further delineated when knockout mice [35] and human males [36] lacking expression of this gene were found to be sterile. Fetal development of the testis in these experiments was not altered. Early embryonic testis morphology in a double knockout remains to be examined [37]. Neonatal exposure to estrogen alters the ERα and ER-β expression during postnatal testis and hypothalamic/pituitary development [38, 39]. Interestingly, the neonatal exposure to the estrogenic compound diethylstilbestrol promotes abnormal testis and male reproductive tract development. Therefore, actions of estrogenic endocrine disruptors on estrogen receptors may impair normal fetal gonadal development or stimulate inappropriate differentiation of cells leading to infertility. Although the estrogen receptors are thought to have a role in testis development [40-42], the specific functions remain to be elucidated. Treatment of males with estrogens during early fetal life may alter responsiveness to androgens by changing androgen receptor (AR) expression patterns [43, 44].
Anti-androgenic endocrine disruptors can also influence fetal gonad development. AR expression is very similar to ER-β expression in the developing testis [32, 45]. However, AR is stage dependent while ER-β expression appears to be more constitutively expressed [32]. AR is detected in Sertoli, myoid, and pre-spermatogonial cells just after cord formation [46]. AR also can be detected in interstitial cells late in development. It is proposed that AR is present in cells that migrate from the mesonephros and enables cord formation to occur [46]. Therefore, inappropriate expression or actions of AR through treatment by endocrine disruptors may effect the process of morphological sex differentiation (cord formation). Anti-androgens such as flutamide [47] or cyproterone acetate (CPA) [48] administered to pregnant rats at different ages of gestation impair fertility in male offspring. Both flutamide and CPA block the ability of androgens and epidermal growth factor (EGF) to stabilize the Wolffian duct [49]. Testosterone has been demonstrated to increase the expression of EGF receptor in the developing testis [49]. Therefore, perturbation of AR may also cause inappropriate expression and action of growth factors in the testis. A commonly used anti-androgenic endocrine disrupter is vinclozolin, which is used as a fungicide in the wine industry [50, 51]. Vinclozolin has been shown to act as an environmental anti-androgen and influence gonad development and fertility.
Methoxychlor is a chlorinated hydrocarbon pesticide currently used in the United States as a replacement for DDT [52]. Methoxychlor can be metabolized by the liver into two demethylated compounds (i.e. mono-OH-M and bis-OH-M). The most active estrogenic metabolite is 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane, (HPTE) [28, 53, 54]. Other methoxychlor metabolites appear to have anti-androgenic activity [27]. HPTE is weakly estrogenic [55-57] and stimulates the expression of estrogen receptors [58]. Recently it has been found that the estrogenic metabolite of methoxychlor HPTE has differential effects on ER-α and ER-β being an ER-α agonist and ER-β antagonist [59, 60]. Other methoxychlor metabolites also have differential effects on ER-α and ER-β [60]. Therefore in examining the actions of methoxychlor or HPTE the ER agonist and antagonist activity needs to be considered, as well as, anti-androgenic activities. Consideration of these differential activities is critical in elucidating the mechanisms of action of endocrine disruptors such as methoxychlor. Previously, methoxychlor metabolites have been shown to act differentially on the ER from different species [61]. The effects of methoxychlor at an embryonic or early postnatal period can influence reproductive functions at later adult periods [62-64]. Neonatal exposure to methoxychlor can influence pregnancy [65, 66], ovarian and hypothalamic function [67, 68], reproductive behavior [69], prostate development [70], thymus development [71], and testis development [72]. Therefore, transient embryonic exposure to an endocrine disruptor can reprogram and/or imprint effects that manifest in the adult on reproductive physiology. A study has shown that effects on a gestating mother may influence subsequent pregnancies as well [73].
Vinclozolin is an anti-androgenic compound that is metabolized into butenoic acid and enanilide derivatives termed M1 and M2, respectively [74]. The affinity of the metabolites for the androgen receptor are 10-15 times (i.e. Ki 10-100 μM) greater than the parent compound [75]. Exposure of neonates to anti-androgenic compounds causes abnormalities in sexual differentiation and gonad formation [75, 76]. Peripubertal exposure to anti-androgens delays puberty, inhibits development of androgen-dependent tissues, and alters androgen receptor function in the male rat [77-79]. Embryonic and early postnatal exposure can influence subsequent male sexual differentiation and fertility [80-83]. The embryonic exposure periods at the time of testis formation appears to be the most sensitive exposure period to the anti-androgens [84]. Evidence with a variety of toxic compounds has determined that metabolites of estrogenic substances such as p,p′DDE (metabolite of DDT) act as anti-androgens and inhibit the transcription of androgen regulated genes [85]. A recent report suggests antagonistic and synergistic interaction effects between vinclozolin and androgens [86].
Thus, the impact of toxic compounds has become more complicated, and their estrogenic and anti-androgenic effects on reproduction and gonadal development need to be investigated.
The adult testis is a complex organ that is composed of seminiferous tubules, which are enclosed by a surrounding interstitium. The seminiferous tubules are the site of spermatogenesis where germ cells develop into spermatozoa in close interaction with Sertoli cells. The Sertoli cells [87] form the seminiferous tubule and provide the cytoarchitectural arrangements for the developing germinal cells [88]. Tight junctional complexes between the Sertoli cells contribute to the maintenance of a blood-testis barrier [89] and create a unique environment within the tubule [90, 91]. The majority of Sertoli secretory products [92-97] are hormonally regulated and provides useful markers of Sertoli cell differentiation. Surrounding the Sertoli cells are a layer of peritubular myoid cells which function in contraction of the tubule. The peritubular cell surrounds and forms the exterior wall of the seminiferous tubule. Peritubular cells are mesenchymally derived cells that secrete fibronectin [98] and several extracellular matrix components [99]. Both the peritubular and the Sertoli cells form the basement membrane surrounding the seminiferous tubule and their interactions are important in germ cell development. The interstitial space around the seminiferous tubules contains another somatic cell type, the Leydig cell that is responsible for testosterone production. Leydig cells have a major influence on spermatogenesis through the actions of testosterone on both the seminiferous tubule and the pituitary. Although, the Leydig cell has numerous secretory products [100], the ability of the cell to produce androgen to act on the seminiferous tubules is the most significant secretory product of the cells. Leydig cell androgen production can be directly influenced by the actions of the endocrine disruptor methoxychlor and its metabolite HPTE [101]. Thus, interaction of all three somatic cells, Sertoli, peritubular and Leydig, are important for regulation of normal spermatogenic function in the testis [100]. The process of fetal testis formation occurs late in embryonic development (embryonic day 13 (E13) in the rat) and is initiated by migration of primordial germ cells, first from the yolk sac to the hindgut and then from the hindgut to the genital ridge. The gonad is bipotential after germ cells migration and morphologically can be distinguished from the adjoining mesonephros (E12 in rat), but cannot be identified as an ovary or a testis. Two events occur early on embryonic day 13 (E13) to alter the bipotential gonad. First, Sertoli cells, which are proposed to be the first cell in the testis that differentiates, aggregate around primordial germ cells [102, 103]. Secondly, migration of mesenchymal cells occurs from the adjoining mesonephros into the developing gonad to surround the Sertoli cell-germinal cell aggregates. The migrating population of cells is speculated to be pre-peritubular cells [104, 105]. The mechanism for this migration is due to a chemotactic signal from the testis to promote cell migration [106]. Therefore, during early testis development Sertoli-peritubular cell interactions promote cord formation to occur. The cords neonatally develop into seminiferous cords and at the onset of puberty develop into the seminiferous tubules. Seminiferous cords form as the Sertoli cell-primordial germ cell aggregates become more organized and are fully surrounded by the migrated mesonephros mesenchymal cells (i.e. pre-peritubular cells). The formation of the seminiferous cords (E14 in rat) is a critical event in the morphogenesis of the testis since this is the first indication of male sex differentiation. During the process of cord formation, the Sertoli cells undergo a number of morphological changes including: a change in expression of mesenchymal to epithelial cell markers (vimentin to cytokeratin), [107] a change in expression of cytokeratin 19 to cytokeratin 18 (cytokeratin 19 is expressed in ovary) [108], and expression of MIS which inhibits the development of the Müllerian duct, the precursor of the female uterus, cervix, fallopian tubes and upper vagina [109, 110]. Outside of the seminiferous cords the peritubular layer of cells become identifiable from the interstitium or Leydig cells at E15 [111] and 3βPHSD production is detected after E15 [112]. This is important since the production of testosterone and androgens by the Leydig cells has been demonstrated to stabilize the Wolffian duct derivatives for normal male duct development [113]. Therefore, appropriate differentiation of somatic cell types in the testis around the time of cord formation is crucial not only for the normal development of the testis, but also for the continued presence of the Wolffian duct.
Primordial germ cells form aggregates with Sertoli cells prior to cord formation [102, 103] and then are localized within the seminiferous cords as testis morphogenesis is initiated. The germ cells undergo a rapid mitosis until the late stages of embryonic development at which time they become quiescent. After birth in the rodent germ cell mitosis resumes and during the onset of puberty and formation of the seminiferous tubules spermatogenesis is initiated. Germ cells throughout development are in close association with the somatic cells (i.e. Sertoli cells). Alteration of somatic cell differentiation could indirectly effect germ cell development, as well as having direct effects on the germ cells.
The testis transcriptome (i.e. global gene expression profile) will change during testis development, due to the differentiation and growth of a variety of different cell types. These changes in the testis transcriptome reflect critical regulatory genes and gene families required to promote normal testis function and development. A variety of functionally related genes such as transcription factors, signal transduction genes, cell cycle genes, cell survival genes, and growth factors will be involved in testis development and part of the transcriptome. The ability of the endocrine disrupter to alter the testis transcriptome and specific genes and gene families is in part one of the mechanisms used to alter fetal and adult testis function and development. Examples of gene families shown to influence embryonic testis development include the epidermal growth factor (EGF) family [114-116], the transforming growth factor β (TGFβ) family [117] and neurotropin growth factor family [118, 119].
Previously it was demonstrated that a transient exposure to methoxychlor or vinclozolin of a gestating mother between embryonic days 8 to 15 (E8-E15) promoted in the adult F1 generation, reduced spermatogenic capacity, increased spermatogenic cell apoptosis and decreased sperm number and motility [120, 121]. The gestating mother rats were injected daily with an intraperitoneal injection of 100 or 200 mg/kg dose of the endocrine disruptor. These doses are similar to previous in vivo studies, and are within anticipated environmental exposure levels [27, 28, 52-72, 74-82, 84, 85, 121-123]. A similar transient exposure between embryonic days 15-20 (E15-E20), had no effect on the F1 generation testis [120, 121].
A number of human diseases are the result of abnormal epigenetic (i.e. DNA methylation) programming including Angelman's syndrome, Beckwin-Wiedemann syndrome and Prader-Willi syndrome [143-145]. Alterations in the DNA methylation patterns of imprinted genes have also been shown to promote the development of disease [146]. Potential epigenetic abnormalities in children from in vitro fertilization (i.e. intracytoplasmic sperm injection) have been identified [147]. A study of monozygotic twins suggested environmental epigenetic effects on disease [148]. Therefore, numerous studies and clinical conditions suggest epigenetics may be a critical factor in disease etiology.
The observation that an environmental toxicant (e.g. endocrine disruptor) can have an epigenetic effect on the germ-line of the gestating mother, and cause a transgenerational affect on male reproduction, significantly impacts our understanding of the hazards of these compounds to human health, as well as all other mammalian species. Elucidation of this phenomenon permits better understanding of the true hazards of environmental toxicants, permits identification of the specific causal agents, and allows the development of appropriate preventative and therapeutic approaches. Independent of the specific compound or agent of interest, the establishment of this potential mechanism of action is critical, and provides insight into the effects of environmental factors that influence embryonic mammalian development and adult reproduction.